International Consensus Guideline for Reporting Transmission Electron Microscopy Results in the Diagnosis of Primary Ciliary Dyskinesia (BEAT PCD TEM Criteria)

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International consensus guideline for reporting transmission electron microscopy results in the diagnosis of Primary Ciliary Dyskinesia (BEAT PCD TEM Criteria)

Amelia Shoemark, Mieke Boon, Christoph Brochhausen, Zuzanna Bukowy-Bieryllo, Maria Margherita De Santi, Patricia Goggin, Paul Griffin, Richard G. Hegele, Robert A. Hirst, Margaret W. Leigh, Alison Lupton, Karen MacKenney, Heymut Omran, Jean-Claude Pache, Andreia Pinto, Finn P. Reinholt, Josep Schroeder, Panayotis Yiallouros, Estelle Escudier

Please cite this article as: Shoemark A, Boon M, Brochhausen C, et al. International consensus guideline for reporting transmission electron microscopy results in the diagnosis of Primary Ciliary Dyskinesia (BEAT PCD TEM Criteria). Eur Respir J 2020; in press (https://doi.org/10.1183/13993003.00725-2019).

This manuscript has recently been accepted for publication in the European Respiratory Journal. It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online.

International consensus guideline for reporting transmission electron microscopy results in the diagnosis of Primary Ciliary Dyskinesia (BEAT PCD TEM Criteria)

Amelia Shoemark1,2, Mieke Boon3, Christoph Brochhausen4, Zuzanna Bukowy-Bieryllo5, Maria Margherita De Santi6, Patricia Goggin7, Paul Griffin1,8, Richard G Hegele9, Robert A. Hirst10, Margaret W Leigh11, Alison Lupton12, Karen MacKenney13, Heymut Omran14, Jean-Claude Pache15, Andreia Pinto16, Finn P Reinholt17, Josep Schroeder4, Panayotis Yiallouros18, Estelle Escudier19 *These authors represent a larger guideline development group acknowledged below. 1. Royal Brompton Hospital, London, UK 2. School of Medicine, University of Dundee, UK 3. Department of Pediatrics, University Hospital Leuven, Belgium 4. Institute of Pathology, University Regensburg, Regensberg, Germany 5. Institute of Human Genetics, Polish Academy of Sciences, Poland 6. Department of Pathology, University Hospital of Siena, Italy. 7. University Hospital Southampton NHS Foundation Trust, Southampton, UK 8. Royal Childrens Hospital, Melbourne, Australia 9. Hospital for Sick Children-University of Toronto, Toronto, Ontario, Canada 10. Department of Respiratory Sciences, University of Leicester, Leicester UK 11. Department of Pediatrics and Marsico Lung Institute, University of North Carolina, Chapel Hill, North Carolina, USA 12. Greater Glasgow and Clyde, Queen Elizabeth University Hospital, Pathology Department, UK 13. NSW Health Pathology, Concord Repatriation General Hospital, Sydney, Australia 14. Department of Pediatrics; University Hospital Muenster; Germany 15. University Hospital of Geneva, Switzerland 16. Instituto de Medicina Molecular, Portugal 17. Oslo University Hospital, Oslo, Norway 18. Medical School, University of Cyprus, Cyprus 19. Sorbonne Université, Faculté de Médecine, INSERM UMR_S933, (APHP) Assistance Publique Hôpitaux de Paris and CHIC (centre hospitalier intercommunal de Créteil), France

ABSTRACT

Primary Ciliary Dyskinesia (PCD) is a heterogeneous genetic condition. European and North American diagnostic guidelines recommend transmission electron microscopy (TEM) as one of a combination of tests to confirm a diagnosis. However, there is no definition of what constitutes a defect or consensus on reporting terminology. The aim of this project was to provide an internationally agreed ultrastructural classification for PCD diagnosis by TEM.

A consensus guideline was developed by PCD electron microscopy experts representing 18 centres in 14 countries. An initial meeting and discussion were followed by a Delphi consensus process. The agreed guideline was then tested, modified and retested through exchange of samples and electron micrographs between the 18 diagnostic centres.

The final guideline a) Provides agreed terminology and a definition of class 1 defects which are diagnostic for PCD; b) Identifies class 2 defects which can indicate a diagnosis of PCD in combination with other supporting evidence; c) Describes features which should be included in a ciliary ultrastructure report to assist multidisciplinary diagnosis of PCD d) Defines adequacy of a diagnostic sample.

This tested and externally validated statement provides a clear guideline for the diagnosis of PCD by TEM which can be used to standardise diagnosis internationally.

INTRODUCTION

Primary Ciliary Dyskinesia (PCD) is a heterogeneous inherited condition affecting 1:10,000 population (1). Symptoms usually begin early in life and include: chronic nasal discharge and wet cough, progressing in childhood to recurrent upper and lower airway infections and eventual bronchiectasis (1). As cilia are also present at the embryonic node defects in nodal cilia may cause abnormalities of left-right laterality determination (situs abnormalities).

Diagnosis of PCD usually relies on a combination of tests, because a single test cannot reliably diagnose all PCD types. These tests may include nasal nitric oxide measurement(2), assessment of ciliary waveform by high speed video microscopy(3), immunofluorescence analysis of ciliary proteins(4), transmission electron microscopy (TEM) and genetic testing ).European and North American diagnostic guidelines differ in recommendations for diagnostic testing although both agree that assessment of ciliary ultrastructure by TEM and/ or bi-allelic mutations in a known PCD gene definitively confirm a PCD diagnosis (5-7). However, there has been considerable heterogeneity among pathology reports describing PCD and interpreting what constitutes a diagnostic defect. Defects of the ciliary axoneme can occur secondary to infection or inflammation of the airway mucosa or in samples which have not been adequately prepared. Differences in reporting and interpretation of findings can lead to misdiagnosis.

In recent years advances in genetic testing and molecular biology have redefined many aspects of PCD, whilst improving understanding of the basic science has further complicated the use of traditional electron microscopy terminology. For example the use of the term ‘radial spoke defect’ has been used to describe both central complex defects such as those caused by RSPH4A mutations and microtubular disarrangement defects caused by CCDC39 mutations (8-11). In other clinical entities such as renal allograft pathology internationally agreed pathology classification known as ‘the Banff criteria’ has been shown to significantly improve diagnostic precision, reproducibility and disease outcome (12, 13). The aim of the present project was to provide an internationally agreed ultrastructural classification for the diagnosis of PCD by defining defects diagnostic for PCD, describing features which should be included in a report to assist multidisciplinary diagnosis of PCD and define adequacy of a diagnostic sample.

METHODS

Guideline development The guideline development process is shown in Figure 1. A group of electron microscopy experts (representing 18 centres in 14 countries: Australia, Belgium, Canada, Cyprus, France, Germany, Italy, Norway, Poland, Portugal, Spain, Switzerland, United Kingdom, USA) met in person and via video link to develop a consensus statement for reporting ciliary ultrastructure by TEM at a COST Action BEATPCD meeting. Minutes from this meeting were used to create common themes and identify items for a Delphi consensus survey. The Delphi consensus survey consisted of three rounds with aims to: a) Define defects diagnostic for PCD

b) Describe features which should be included in a report to assist multidisciplinary diagnosis of PCD c) Define adequacy of a diagnostic sample

Agreement was considered to be met if > 80% of the participants agreed. Consultation with the wider BEATPCD community was conducted at the next BEATPCD meeting.

Guideline validation

Once a draft guideline was complete a series of TEM sections were distributed among the group and the guideline was tested and modified for inconsistencies and clarity.

A list of 16 cases representing patients that had a genetically confirmed diagnosis of PCD or conclusively did not have PCD were used for the guideline validation. Four centres were assigned four cases from the following to prepare: CCDC114, CCDC39, CCDC40, CCNO, DNAAF1, DNAAF3, DNAH5, DNAH11, DRC1, RSPH4A, cystic fibrosis, healthy volunteer (x2), inadequate sample (x2), unaffected sibling of a patient with PCD. Centres at Southampton (UK), Toronto (Canada), Paris (France) and Muenster (Germany) sectioned and stained 4 grids from 4 cases each. These grids were sent to London where they were quality checked, anonymised, randomised and distributed by an individual who was not part of the consensus group. 17 centres then received 4 grids and 1 centre images. Centres did not receive their own grids.

All 17 centres assessed the 4 grids, each centre using its own technology for making and interpreting images. The 18th centre received images. The centres completed a report form based on the consensus statement and returned representative images. The form included class of defect, ultrastructural defect, adequacy of the sample and a summary of key findings. One centre reviewed images taken at the distribution centre in London, rather than grids, due to technical difficulties.

Results of the validation exercise were discussed in a face to face meeting of the group. One additional online survey was distributed to clarify points from this meeting and the guideline was modified accordingly. Results of the guideline surveys and validation can be found in the supplementary information.

Finally images from the cases with CCDC39, DNAAF3, DNAH5, RSPH4A, and cystic fibrosis were used to re-assess the modified completed guideline. This validation exercise was completed by all consensus statement authors and 6 additional electron microscopists who were not directly involved in developing the consensus statement.

RESULTS

This guideline describes assessment and reporting of ciliated epithelial samples fixed in glutaraldehyde, dehydrated, embedded in resin and stained with heavy metals (lead citrate and uranyl acetate or equivalent).

Normal ultrastructure of respiratory cilia. The consensus group identified that it is crucial for the observer to be familiar with the appearance of normal ciliary ultrastructure and PCD defects from extensive samples using local sampling, processing and visualisation methods before embarking on diagnostic testing for PCD. Figure 2 shows the normal ultrastructure of a healthy respiratory cilium. The

axoneme of motile cilia is composed of the well-known “9+2” structure: 9 peripheral microtubule doublets surround the central pair of single microtubules. Each microtubular doublet is connected with the central pair by radial spokes, and neighboring MT doublets are connected by the nexin dynein regulatory complex (N-DRC). The outer microtubular doublets contain regularly repeating hook shaped structures known as outer dynein arms (ODA) and inner dynein arms (IDA), which are responsible for the generation of ciliary motion through ATPase activity. Essential units repeat along the axoneme approximately every 96nm. Each doublet contains in a 96 nm repetition 4 double headed outer dynein arms (ODAs), 6 different single headed and 1 double headed inner dynein arm (IDA) isoform, 1 N-DRC and 3 radial spokes (14-17). Differences in the repetition of these units and their density results in differing contrasts on TEM. The electron dense ODA is easier to visualise than the less dense IDA and complex-DRC in a 70-100nm TEM section, because within the slice more ODA dynein heavy chains are present. Ultrastructure at the tip and base of the cilium is different and this should be considered when assessing for PCD defects.

Class 1 and class 2 defects, terminology, adequacy of sampling and consensus definitions for the diagnosis of PCD by TEM

The expert consensus group identified two classes of PCD TEM diagnostic defects: Class 1 defects which are considered ‘hallmark’ defects confirming a diagnosis in a patient with symptoms of the condition and class 2 defects which indicate a diagnosis of PCD in a patient with clinical symptoms of the condition if consistent across more than one sample or after cell culture AND consistent with other test results. The two classes of defects are shown in table 1 and defined in the text. The group made distinction between the two classes of defect as class one defects are diagnostic whereas class two defects can be more difficult to recognise and can be similar to secondary defects.

Table 1: Summary of class 1 and class 2 defects for the ultrastructural diagnosis of primary ciliary dyskinesia

Class 1 defects: Hallmark diagnostic defects  Outer dynein arm defect  Outer and inner dynein arm defect  Microtubular disorganisation and inner dynein arm defect

Class 2 defects: Indicate a PCD diagnosis with other supporting evidence  Central complex defect  Mislocalisation of basal bodies with few or no cilia  Microtubular disorganisation defect with inner dynein arm present  Outer dynein arm absence from 25%-50% cross sections  Combined inner and outer dynein arm absence from 25-50% cross sections

Class 1 defects (hallmark defects)

Description: Class 1 defects are diagnostic of PCD in a patient with clinical symptoms of the condition. Sample adequacy: Class 1 defects are confirmed following assessment of >50 axonemes in transverse section from several cells. Care should be taken to assess cross sections from both proximal (in the

region of the microvili) and distal regions of the axoneme. Dynein arms should be assessed in cross sections with clear structural features and an intact ciliary membrane. Microtubular arrangement may be assessed in a larger number of cross sections with an intact membrane in which the dynein arms may not be clear. Examples of class 1 defects are shown in Figure 3.

Outer dynein arm (ODA) defect:

Definition: Absence of the whole or larger part of the outer dynein arm structure from the majority of (> 5) microtubular doublets in the majority (>50%) of cilia cross sections

The defects arise from mutations in genes coding for ODA structural proteins (e.g DNAH5) and ODA docking proteins (e.g.CCDC151). IF analyses usually show an absence of the outer dynein heavy chain protein DNAH5.

Outer and inner dynein arm (ODA+IDA) defect:

Definition: Absence of the whole or larger part of the outer dynein arm structure from the majority of (> 5) microtubular doublets in the majority (>50%) of cilia cross sections AND absence of the whole or larger part of the inner dynein arm structure from the majority of (> 7) microtubular doublets in the majority (>50%) of cilia cross sections.

If the number of inner dynein arms cannot be accurately determined samples should be reported as outer dynein arm defect (+/- inner dynein arm defect)

These defects often arise from mutations in dynein assembly genes. IF analyses show absence of proteins such as DNAH5 and DNALI1.

Microtubular disorganisation and inner dynein arm defect:

Definition: Disruption of the 9+2 symmetry of the microtubules in >25% cross sections combined with absence of the whole or larger part of the inner dynein arm structure from the majority of (> 7) microtubular doublets in the majority (>50%) of cilia axonemes visualised in cross section.

The defects almost always arise from CCDC39 or CCDC40 mutations.7 IF analyses show in those cases absence of N-DRC proteins such as GAS8 and inner dynein arm proteins such as DNALI1.

Class 2 defects

Description: Class 2 defects can indicate a diagnosis of PCD in a patient with clinical symptoms of the condition if consistent across more than one sample AND consistent with other results such as those from immunofluorescence, high speed video microscopy or genetic analysis. They may require assessment of more ciliary axonemes than class 1 defects. Care should be taken to assess cross sections from several cells and both proximal (in the region of the microvili) and distal regions of the axoneme. Dynein arms should be assessed in cross sections with clear structural features and an intact ciliary membrane. Microtubular arrangement may be assessed in a larger number of cross sections with an

intact membrane in which the dynein arms may not be clear. Examples of class 2 defects are shown in Figure 4.

Central complex defect:

Description: Central complex defects consist of a proportion of normal cross sections and consistent cross sections with one or both of the central microtubule(s) absent (usually >20%) Occasional double central pairs or translocation of the outer microtubule to the central region (8+1) may be seen. Translocation typically occurs approaching the ciliary tip. In longitudinal sections, Intermittent or complete loss of the central pair, or transposition of an outer doublet may also be seen. Some microtubular disorganisation is often seen. These TEM findings are reported to be associated with genetic defects of the radial spoke components (e.g. RSPH4A, RSPH1, RSPH9 and DNAJB13)(18-20) which can be confirmed by immunofluorescence for radial spoke head proteins. In some cases high speed video microscopy can show circling of cilia when observed from above.

Mislocalisation of basal bodies with few or no cilia:

Description: Findings are typically of no or very few ciliary cross sections in combination with failure of the majority of basal bodies to dock at the apical surface of the cell, meaning they are consistently seen within the cytoplasm. These findings are associated with genetic variants in CCNO and MCIDAS and by high speed video microscopy only a few cilia are seen per cell (21, 22). The genetic defects result in reduced generation of multiple motile cilia. In the rare cilia that are visualised the ultrastructure of the axoneme in CCNO cases can be normal but in individuals with MCIDAS mutations can lack ODAs.

Microtubular disorganisation:

Description: Disruption of the 9+2 symmetry of the microtubules in cross sections consistently throughout an otherwise healthy sample. The absence of the dynein regulatory complex/nexin link is sometimes noted. Usually the majority of cross sections have normal ultrastructure with outer and inner dynein arms present. These findings might be associated with mutations in CCDC65, DRC1 and GAS8 encoding N-DRC proteins (23-25). Subtle beat pattern abnormalities including fast or disrupted beat pattern and an absence of N-DRC proteins such as GAS8 are observed by immunofluorescence in these cases. The inner dynein arm is present in >50% of cross sections, distinguishing this from the class1 microtubular disorganisation and inner dynein arm defect.

Outer dynein arm absence from 25%-50% cross sections

This definition is the same as for a class 1 outer dynein arm defect except dynein arm(s) are absent in a minority of cilia and numerous normal ciliary cross sections are present. A pattern to the defect may be observed e.g. In individuals with recessive DNAH9 mutations the distal ciliary axonemes lack ODAs whereas the proximal ciliary axonemes are normal. (26, 27). Where a pattern to the defect is observed by TEM immunofluorescence may also show a partial absence of DNAH5.

Combined inner and outer dynein arm absence from 25-50% cross sections

This definition is the same as for a class 1 outer and inner dynein arm defect except for the dynein arms being absent in a minority of cilia and numerous normal ciliary cross sections are present. Some static cilia may be present by high speed video microscopy and immunofluorescence may or may not show a partial absence of DNAH5 and DNALI1. This is seen for example in patients with the missense mutation His154Pro in CCDC103 (28, 29).

The transmission electron microscopy cilia report

A TEM report should conform to general considerations for good practice in pathology reporting:

Reports should be clear and accurate and the overall result or conclusion must be clearly visible. Patients must be identified on reports by at least two unique items of information e.g. full name and date of birth. It must be stated if the testing is incomplete and/or where the minimum quality is not achieved. For PCD testing it should be made clear to the person receiving the report that normal ultrastructure does not exclude a diagnosis.

Table 2 describes items agreed by the expert group as essential and desirable for an electron microscopy report in the diagnosis of PCD

Table 2: Items to include in a TEM report for the diagnosis of Primary Ciliary Dyskinesia

Essential items to be included in a TEM report for PCD  Source of the sample (e.g. nasal brushing)  Adequacy of the sample  Number of cross sections assessed  % abnormal cilia (class 1 and class 2 defects)  Consistency of a defect across several cells  One sentence summary of key findings (including class 1 or 2 defect if present)

Additional items to consider enhancing a report  Orientation/ alignment of the basal body or central pair of microtubules  Number of cells assessed  Blebs/membrane swelling/membrane condition  Presence of compound cilia (more than one axoneme within a membrane)  Preservation of the sample  % cilia with other defects  Presence of shortened or truncated ODA projections  Evidence of inflammation  Evidence of bacteria

Summary of guideline development survey results (Full results are shown in the supplementary information) In round 1 consensus was met on the 3 hallmark defects shown in table 1 and participants suggested definitions. >80% agreed that the following were class 2 defects: Central complex defect, Mislocalisation of basal bodies with few or no cilia, Outer dynein arm absence from 25%-50% cross sections, Combined inner and outer dynein arm absence from 25-50% cross sections. Microtubular disorganisation was added to this list following the validation exercise. Items to include in a report were also agreed (table 2).

The consensus group did not reach consensus on the following items as class 1 or 2 defects and inclusion or exclusion of these should be at the discretion of the observer as evidence becomes available: Isolated inner dynein arm defects (30), ODA loss <25% cross sections, ODA and IDA loss <25% cross sections, orientation defect (31-33), ciliary length defects. Future research should focus on the relevance of these defects and the guideline should be updated accordingly.

In round 2 and 3 definitions of hallmark defects were agreed along with the criteria for sample adequacy, i.e. >50 cross sections with intact ciliary membranes.

Summary of guideline validation results

Two rounds of validation were conducted. There was 100% participation (18 centres) in this activity. In round one there were no false positive diagnoses (i.e no non-PCD sample was classed as having a Class 1 Hallmark PCD defect). 17/68 (25%) sections were described as insufficient or inadequate for assessment. These included 100% returns on a CCNO case and 75% returns for a sample included as an inadequate sample. 25/25 cases (100%) were correctly identified as a class 1 hallmark defect. However 8/25 (32%) returns recorded an incorrect name of the class 1 defect. 2 cases were described as having a class 1 defect when they had a class 2 defect or normal ultrastructure. 5/6 cases were correctly identified as class 2 defects. 4 normal ultrastructure cases were erroneously identified as a class 2 defect.

The guideline was modified and in round two there were no false positive diagnoses (i.e. no non-PCD sample was classed as having a Class 1 hallmark PCD defect) and normal ultrastructure identified by 18/18 centres. There were no false negatives. There was 100% correct identification as a class 1 hallmark defect. These included 18/18 correctly identified MTD + IDA, 17/18 ODA (1 judged as ODA+IDA) and 17/18 ODA+IDA (1 judged ODA). There was 100% correct identification as a class 2 defect. 15/18 correctly identified a central complex defect whereas 3 judged this as MTD. This stresses the importance of the additional evidence for class 2 defects. 94% of reviewers gave a correct ultrastructural defect according to the genetic diagnosis. Agreement between observers was kappa 0.76 -1.0 (where 0= no agreement and 1= complete agreement).

For external validation a further 6 electron microscopists were given the guideline to read and then completed the image validation. All six observers identified the correct ultrastructural defect for all cases.

Discussion

Next generation sequencing and molecular advances have resulted in fast paced improvement of PCD diagnosis over recent years. TEM is valuable to assert and/or to confirm a diagnosis of PCD and as a guide for genetic testing, as there is a clear link between the ultrastructural defect and the group of affected genes. This is useful especially when variants of unknown significance are identified. Pathogenic mutations in known PCD genes currently only account for 65-75% cases of PCD confirmed by TEM. A table of known TEM phenotypes according to genotype is available in the supplementary information. We present the first internationally agreed guideline for reporting of ciliary biopsies for the diagnosis of PCD. This guideline is designed to be a flexible formulation, it will require updating and evolution as new evidence becomes available. These recommendations are minimum requirements and professional judgement and experience is of paramount importance in many circumstances.

PCD with normal ultrastructure

It has been shown repeatedly that the diagnostic accuracy of TEM is not sufficient to exclude a diagnosis as up to 30% of all PCD cases are described as having normal or near normal ciliary ultrastructure (34- 36). This includes the large dynein heavy chain DNAH11 which accounts for a substantial number of cases (37, 38). In another group of patients subtle changes to ultrastructure can be seen but alone these are non-diagnostic for example defects in HYDIN which affect the c2b projection of the central pair (39). The current statement introduces the concept of class 2 defects to address limitations with the identification of subtle changes. Class 2 defects support a diagnosis but should not be used without other diagnostic tests. Many of these class 2 defect changes are secondary to the causative molecular defect, for example radial spoke head gene defects result in secondary loss of the central pair (19). It must be stressed that some class 2 defects, if not confirmed in a second sample or after cell culture, may also be the result of non-PCD related causes.

Limitations of electron microscopy in the diagnosis of PCD

Initial validation of the guideline (supplementary information) revealed that smaller inadequate samples were most likely to lead to a misdiagnosis. It was also clear that individual processing techniques and familiarity with local appearance of cilia is paramount to interpretation. Due to differences in equipment availability and local procedures it is not currently possible to standardise all methodology in sample processing and visualisation. In the future a consensus methodology could be developed. However we have addressed methodological differences by recommending that extensive normal and PCD samples are visualised using local techniques before a diagnostic sample is interpreted.

Secondary ultrastructural defects in cilia can be caused by acute or chronic respiratory infections, inflammatory respiratory disease (i.e. asthma), environmental and demographic factors (smoking, pollution, age) or sample handling (40). It is common to observe secondary defects in samples examined by TEM, for example microtubular defects that have been observed in up to 10% of healthy controls (41, 42). The appearance of secondary defects can overlap with the appearance of positive PCD cases e.g., microtubular disorganisation, central pair abnormalities. Misinterpretation of these defects can be prevented by taking care to avoid sampling when the patient is unwell, use of cell culture of respiratory epithelium (air-liquid interface or spheroid cultures) to remove conditions causing the

secondary effects, consideration of the health of individual cells in the sample and by repeating the biopsy(43, 44). To ensure that occasional secondary defects in the sample do not bias interpretation, we recommend assessment of at least 50 axonemes in transverse section from a number of different cells. We recommend dynein arms should be assessed in cross sections with clear structural features and intact ciliary membranes. Microtubular arrangement may be assessed in a larger number of cross sections with intact membranes in which the dynein arms are not clear although unhealthy cells or compound cilia should be avoided. For class 2 defects we recommend assessment of more than one sample or following cell culture. Paramount is the consistency of a class 2 defect with results of other tests such as high speed video, immunofluorescence analysis, nasal nitric oxide measurement and genotyping.

Summary

Performance (sensitivity and specificity) of TEM for PCD diagnosis is difficult to accurately assess when there is no agreed diagnostic criteria. This consensus statement is tested and validated and provides a clear guideline for the diagnosis of PCD by TEM which can be used to standardise diagnosis internationally.

Supplementary information:

Results of consensus surveys

Results of validation activities

Example cases and reports for hallmark and class 2 defects

Table of genotype associations with TEM phenotype

Figures

Figure 1: Flow chart outlining the methodology for development of the TEM consensus guideline. Results of Delphi surveys and case reviews can be found in the supplementary information

Figure 2: Electron micrographs showing normal ciliary ultrastructure in cross section A) The core of the ciliary axoneme with 9+2 microtubular arrangement. Black arrows depict the outer dynein arms (ODA) and black arrows with white outline the inner dynein arm (IDA). B) Cross sections form the tip of the cilium with single microtubules (indicated by grey arrows) are a normal part of ciliary ultrastructure and should not be assessed for diagnosis of class 1 or class 2 defects. C) Ultrastructurally normal ciliary cross sections from the base of the axoneme. White arrow indicates that these cross sections have no central pair. Microtubular doubles are often linked to the ciliary membrane with Y shaped linker D) Diagrammatic representation of ciliary cross sections at the tip, central axoneme and base

Figure 3: Electron micrographs and diagrams of class 1 ultrastructural defects in cross section. Left panel; Outer dynein arm defect, central panel outer and inner dynein arm defect, right panel microtubular disorganisation and inner dynein arm defect. Please note not all arms are completely missing. Although the curved hook structure of the ODA is not present in its entirety the white star indicates some remaining proximal structure likely to be the docking complex. Class 1 defects are diagnostic of PCD in patients with symptoms of the condition. Class 1 defects are confirmed following assessment of >50 axonemes in transverse section. Dynein arms should be assessed in cross sections with clear structural features and an intact ciliary membrane as depicted. Microtubular arrangement may be assessed in a larger number of cross sections with an intact membrane in which the dynein arms may not be clear.

Figure 4: Electron micrographs of class 2 ultrastructural defects. Left panel Central complex defect with features A-D demonstrating, lack of central pair, transposed outer microtubular doublet, single central tubule and double central pair. Micrograph from an individual with an RSPH4A mutation. Right panel Mislocalisation of basal bodies with few or no cilia from an individual with a CCNO mutation

One representative lead author (usually the individual assessing and reporting the TEM and representing the opinions of each centre by completing the Delphi survey) per centre has been included in the authorship of this report. However, several additional experts have attended meetings, commented on this consensus statement and contributed their expert opinion and technical skills during it’s development. We would like to thank: Sverre-Henning Brorson, Tom Burgoyne, Suzanne Crowley, Chris O’Callaghan, Mellisa Dixon, Regan Doherty, Catherine Faucon, Paul French, Raffaella Guazzo, Yew Meng Heng, Claire Hogg, Merete Helgesen, Louise Hughes, Martine Jaspers, Panayiotis Kouis, Kyriacou Kyriacos, Jane Lucas, Virginia Mancini. Paul Martinello, Rana Mitri, Elena Moretti, José Moura Nunes, Anton Page, Phil Robinson, Anna Rowinska , Andrew Rutman, Heiko Siegmund, Jennifer Sweeney, James Thompson, and Valerie Vlaeminck.

In addition we are especially grateful to Farheen Dauvohra for anonymising and randomising sections and collating data for the validation of this guideline

This project was conducted as part of BEAT-PCD, a network of multidisciplinary researchers and clinicians funded by EU COST Action (BM1407)

References

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19. Castleman VH, Romio L, Chodhari R, Hirst RA, de Castro SC, Parker KA, et al. Mutations in radial spoke head protein genes RSPH9 and RSPH4A cause primary ciliary dyskinesia with central-microtubular- pair abnormalities. Am J Hum Genet. 2009;84(2):197-209. 20. El Khouri E, Thomas L, Jeanson L, Bequignon E, Vallette B, Duquesnoy P, et al. Mutations in DNAJB13, Encoding an HSP40 Family Member, Cause Primary Ciliary Dyskinesia and Male Infertility. Am J Hum Genet. 2016;99(2):489-500. 21. Boon M, Wallmeier J, Ma L, Loges NT, Jaspers M, Olbrich H, et al. MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia. Nat Commun. 2014;5:4418. 22. Wallmeier J, Al-Mutairi DA, Chen CT, Loges NT, Pennekamp P, Menchen T, et al. Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia. Nat Genet. 2014;46(6):646-51. 23. Olbrich H, Cremers C, Loges NT, Werner C, Nielsen KG, Marthin JK, et al. Loss-of-Function GAS8 Mutations Cause Primary Ciliary Dyskinesia and Disrupt the Nexin-Dynein Regulatory Complex. Am J Hum Genet. 2015;97(4):546-54. 24. Horani A, Brody SL, Ferkol TW, Shoseyov D, Wasserman MG, Ta-shma A, et al. CCDC65 mutation causes primary ciliary dyskinesia with normal ultrastructure and hyperkinetic cilia. PLoS One. 2013;8(8):e72299. 25. Wirschell M, Olbrich H, Werner C, Tritschler D, Bower R, Sale WS, et al. The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans. Nat Genet. 2013;45(3):262-8. 26. Fassad MR, Shoemark A, Legendre M, Hirst RA, Koll F, le Borgne P, et al. Mutations in Outer Dynein Arm Heavy Chain DNAH9 Cause Motile Cilia Defects and Situs Inversus. Am J Hum Genet. 2018;103(6):984-94. 27. Loges NT, Antony D, Maver A, Deardorff MA, Gulec EY, Gezdirici A, et al. Recessive DNAH9 Loss- of-Function Mutations Cause Laterality Defects and Subtle Respiratory Ciliary-Beating Defects. Am J Hum Genet. 2018;103(6):995-1008. 28. Shoemark A, Moya E, Hirst RA, Patel MP, Robson EA, Hayward J, et al. High prevalence of CCDC103 p.His154Pro mutation causing primary ciliary dyskinesia disrupts protein oligomerisation and is associated with normal diagnostic investigations. Thorax. 2017. 29. Panizzi JR, Becker-Heck A, Castleman VH, Al-Mutairi DA, Liu Y, Loges NT, et al. CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms. Nat Genet. 2012;44(6):714-9. 30. O'Callaghan C, Rutman A, Williams GM, Hirst RA. Inner dynein arm defects causing primary ciliary dyskinesia: repeat testing required. Eur Respir J. 2011;38(3):603-7. 31. Bukowy-Bieryllo Z, Zietkiewicz E, Loges NT, Wittmer M, Geremek M, Olbrich H, et al. RPGR mutations might cause reduced orientation of respiratory cilia. Pediatr Pulmonol. 2013;48(4):352-63. 32. Jorissen M, Willems T. The secondary nature of ciliary (dis)orientation in secondary and primary ciliary dyskinesia. Acta Otolaryngol. 2004;124(4):527-31. 33. Rutman A, Cullinan P, Woodhead M, Cole PJ, Wilson R. Ciliary disorientation: a possible variant of primary ciliary dyskinesia. Thorax. 1993;48(7):770-1. 34. Boon M, Smits A, Cuppens H, Jaspers M, Proesmans M, Dupont LJ, et al. Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure. Orphanet J Rare Dis. 2014;9:11. 35. Knowles MR, Daniels LA, Davis SD, Zariwala MA, Leigh MW. Primary ciliary dyskinesia. Recent advances in diagnostics, genetics, and characterization of clinical disease. Am J Respir Crit Care Med. 2013;188(8):913-22.

36. Kouis P, Yiallouros PK, Middleton N, Evans JS, Kyriacou K, Papatheodorou SI. Prevalence of primary ciliary dyskinesia in consecutive referrals of suspect cases and the transmission electron microscopy detection rate: a systematic review and meta-analysis. Pediatr Res. 2017;81(3):398-405. 37. Bartoloni L, Blouin JL, Pan Y, Gehrig C, Maiti AK, Scamuffa N, et al. Mutations in the DNAH11 (axonemal heavy chain dynein type 11) gene cause one form of situs inversus totalis and most likely primary ciliary dyskinesia. Proc Natl Acad Sci U S A. 2002;99(16):10282-6. 38. Knowles MR, Leigh MW, Carson JL, Davis SD, Dell SD, Ferkol TW, et al. Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure. Thorax. 2012;67(5):433-41. 39. Olbrich H, Schmidts M, Werner C, Onoufriadis A, Loges NT, Raidt J, et al. Recessive HYDIN mutations cause primary ciliary dyskinesia without randomization of left-right body asymmetry. Am J Hum Genet. 2012;91(4):672-84. 40. Dixon M, Shoemark A. Secondary defects detected by transmission electron microscopy in primary ciliary dyskinesia diagnostics. Ultrastruct Pathol. 2017;41(6):390-8. 41. de Iongh RU, Rutland J. Ciliary defects in healthy subjects, bronchiectasis, and primary ciliary dyskinesia. Am J Respir Crit Care Med. 1995;151(5):1559-67. 42. Rossman CM, Lee RM, Forrest JB, Newhouse MT. Nasal cilia in normal man, primary ciliary dyskinesia and other respiratory diseases: analysis of motility and ultrastructure. Eur J Respir Dis Suppl. 1983;127:64-70. 43. Jorissen M, Willems T, Van der Schueren B, Verbeken E. Secondary ciliary dyskinesia is absent after ciliogenesis in culture. Acta Otorhinolaryngol Belg. 2000;54(3):333-42. 44. Hirst RA, Jackson CL, Coles JL, Williams G, Rutman A, Goggin PM, et al. Culture of primary ciliary dyskinesia epithelial cells at air-liquid interface can alter ciliary phenotype but remains a robust and informative diagnostic aid. PLoS One. 2014;9(2):e89675.

Supplementary Information

Contents Supplementary Information ...... 1 1.0 Delphi Survey results ...... 1 1.1 Results from 1st survey ...... 1 1.2 Results from 2nd survey...... 2 1.3 Results from final survey ...... 3 2.0 Summary of consensus validation analysis ...... 4 2.1 1st validation summary - grid swap ...... 5 2.2 2nd validation summary - photograph swap ...... 5 3.0 Link to example images...... 5 4.0 Table of Genotype by TEM phenotype ...... 6 5.0 Supplementary Figure 1……………………………………………………………………………………………………………………………………………….7

6.0 Example reports……………………………………………………………………………………………………………………………………..……………………7

1.0 Delphi Survey results

1.1 Results from 1st survey Question 1: Please select which of the following you consider to be a hallmark PCD defect. We unanimously agreed that these three defects are hallmark: · Outer dynein arm absence · Combined outer and inner dynein arm absence · Combined inner dynein arm absence and microtubular disorganisation.

Question 2: Would you consider any of the following TEM defects to be diagnostic of PCD?

We agreed (>80%) that the following defects can be considered to be diagnostic of PCD but not always (i.e they are not hallmark)

Central complex defect Mislocalisation of basal bodies with few or no cilia Outer dynein arm absence from 25%-50% cross sections Combined inner and outer dynein arm absence from 25-50% cross sections

We did not reach consensus regarding the following: Inner dynein arm defect (2 did not respond): No 43% Yes (any category) 57% Isolated microtubular disorganisation (1 did not respond): No 29% Yes (any category) 71% Outer dynein arm absence <25% cilia (1 did not respond): No 21% Yes (any category) 79% Combined outer and inner dynein arm absence <25% cilia (1 did not respond): No 21% Yes (any category) 79% 1

Orientation defect: No 43% Yes (any category) 57% Abnormally long cilia: No 57% Yes (any category) 43%

Question 3: Please describe what you consider to be 'hallmark 'for each of the following e.g. 'outer dynein arm absence': absence of the whole outer dynein arm from the majority of cross sections.

We have many descriptions of the hallmark defects. Highlighting nicely why this consensus is necessary. These have been combined together for example for the ODA with the phrase below for voting in the second round.

Absence of the whole or part of the outer dynein arm structure from the majority of (>5) microtubular doublets in the majority (>50%) of cilia cross sections OR Presence of the complete outer dynein arm structure on the minority of (≤ 4) microtubular doublets in the majority (>50%) of cilia cross sections.

Question 4: Which of the following items is it important to include in a TEM cilia report? We met consensus (>80%) that it is important to include in every TEM cilia report · Source of the sample (e.g. nasal brushing) · Adequacy of the sample · The number of cross sections assessed · % abnormal cilia · Presence of the central complex · The consistency of a defect across several cells · 1 sentence summary of key findings

We also met consensus that in (all or some) circumstances the following should be included: · Orientation/ alignment of the basal body or central pair of microtubules · The number of cells assessed · Blebs/membrane swelling/membrane condition · Presence of compound cilia (more than one axoneme within a membrane) · Preservation of the sample · % cilia with a hallmark defect · Presence of shortened or truncated ODA projections · Microtubular organisation · Evidence of inflammation · Evidence of bacteria

We did not meet consensus on · Estimated location of defect (distal or proximal) · location of the basal body · Presence of nexin links/DRC · Presence of radial spokes · Fixation and processing protocol · Section thickness · Comments on the semi- thin sections · Evidence of blood · % ciliated cells · Length of cilia

1.2 Results from 2nd survey Question 1: Please state whether you agree or disagree with the definitions in the text above. If you disagree please rephrase the definition in the comments box below

18 respondents:

ODA defect definition 17/18 agreed (94%)

ODA+IDA defect definition 13/18 agreed (72%) DID NOT MEET CONSENSUS

IDA & MTD defect definition 15/18 agreed (83%)

Question 2: Using the definition above what is the minimum number of ciliary axonemes in cross section needed to assess before confirming a hallmark defect

Consensus 80% = 50, median = 50, mode = 50, mean = 58

Question 3: Please list your criteria for inclusion of a ciliary axoneme in cross section in your assessment. Examples may include: an intact membrane, sufficient contrast to see the radial spokes, visualisation of a healthy epithelial cell from which the cross section originates, presence of 9 microtubular doublets and a central pair etc.This question is designed to understand differences in the number of cross sections we assess. Themes included: Membrane =14, Cell =6, Contrast =5, Visulisation of other structures =10 Question 4: The following defects can be considered to be suggestive of PCD if reproducible on a second sample or culture AND consistent with supporting evidence from other investigations

We met consensus on Central complex defect (83%) Outer dynein arm absence from 25-50% cilia (83%) Combined outer dynein arm and inner dynein arm absence from 25-50% cilia (82%)

We did not meet consensus Microtubular disorganisation with IDA present (78%)

Mislocalisation of basal bodies with few or no cilia (77%) Outer dynein arm absence from <25% cilia (50%)

Outer and inner dynein amr absence <25% cilia (50%)

1.3 Results from final survey

Q1: Please state whether you agree or disagree with the definitions in the text box above.

Inner and outer dynein arm defect: Absence of the whole or larger part of the outer dynein arm structure from the majority of (> 5) microtubular doublets in the 3 majority (>50%) of cilia cross sections coupled with absence of the whole or larger part of the inner dynein arm structure from the majority of (> 7) microtubular doublets in the majority (>50%) of ciliary axonemes visualised in cross section

Agree 82% Q2: In the first round of the survey we agreed '% defects' should be included in the report. In the second round discussions we realised this is a vague definition. Do you agree or disagree the following should be included in the final TEM report

% Hallmark defects Agree 94% % Hallmark and class 2 defects Agree 82%

% All defects Agree 65% 2.0 Summary of consensus validation analysis

2.1 1st validation summary - grid swap Diagnosis Expected guideline result Returned results 1 2 3 4 5 6 DNAI2 Class 1: ODA ODA+IDA ODA Insuff ODA DNAAF1 Class 1: ODA+IDA ODA+IDA ODA+IDA ODA+IDA ODA+IDA Cystic fibrosis Normal Ultrastructure Normal Ultrastructure Normal Ultrastructure Normal Ultrastructure (chronic bronchitis) Normal Ultrastructure Insuff Normal Ultrastructure DRC1 Normal Ultrastructure (MTD) Normal Ultrastructure (MTD) Normal Ultrastructure (MTD) Normal Ultrastructure Normal Ultrastructure (MTD) Insuff Normal Ultrastructure CCNO Class 2: mislocalised BB + few or no cilia Insuff Insuff Insuff Insuff CCDC40 Class 1: IDA + MTD IDA + MTD Insuff IDA + MTD IDA + MTD CCDC114 Class 1: ODA ODA+IDA ODA+IDA ODA Mixed (predominant ODA) Inadequate - non pcd Inadequate Insuff Insuff Class 2: mislocalised BB + few or no cilia Insuff RSPH4A Class2: CC IDA + MTD CC CC CC CC CC DNAAF3 Class 1: ODA+IDA ODA+IDA ODA+IDA ODA ODA+IDA CCDC39 Class 1: IDA + MTD insuff (IDA+MTD) ODA Insuff Insuff Healthy Volunteer Normal Ultrastructure Insuff class 2 Normal Ultrastructure Normal Ultrastructure DNAH11 Normal Ultrastructure ODA Insuff Normal Ultrastructure Normal Ultrastructure DNAH5 Class 1: ODA ODA ODA+IDA ODA+IDA ODA+IDA ODA ODA Inadequate - Unaffected sibling Inadequate class 2 lack cilia Insuff Insuff CC Main results summarised

1. 100% participation 2. There were no false positive diagnoses (i.e No non-PCD sample was classed as having a Class1 Hallmark PCD defect) 3. 17/68 (25%) sections were described as insufficient or inadequate for assessment a. These included 100% returns on a CCNO case and 75% returns for a sample included as an inadequate sample. The 4th operator defined this inadequate sample as class 2: basal body mislocalisation with few or no cilia. b. In 2 further samples in which75% returns recorded mostly as inadequate the 4th operator recorded the incorrect defect.

4. 25/25 (100%) correct identification as a class 1 hallmark defect a. However 8/25 (32%) returns recorded an incorrect name of the class 1 defect i. ODA defect: recorded as ODA+IDA defect (n=6) ii. ODA and IDA defect: recorded as ODA defect (n=1) iii. MTD and IDA defect: recorded as ODA defect (n=1) b. 2 cases were described as having a class1 defect when they had a class 2 defect or normal ultrastructure i. 1 DNAH11 case described as ODA defect ii. 1 RSPH4a case descried as IDA + MTD

5. 5/6 correct identification of class 2 defects 6. 4 normal ultrastructure cases identified as a class 2 defect a. 2 central complex

b. 1 mislocalisation of basal bodies

Comments, feedback and suggested actions for discussion

All centres have received feedback on individual results and asked for feedback on the process and guideline. If you have not please let me know

Inadequate or insufficient samples

- There were some patterns as to which were deemed inadequate but not all can be explained by poor quality grids. Reasons listed in feedback included: Poor contrast, poor orientation, insufficient cilia, different grid types, different types of sample e.g. culture vs biopsy vs brushing. Operators may find it more difficult to assess grids which were not prepared at their own centre.

*Action: Following update of the guideline re-assess using TEM photographs of cross sections

- Some reports described samples with microtubular defects as inadequate because microtubules could not clearly be seen.

*Action: Update guideline to stress the importance of assessment of arms in perfect cross sections but microtubular organisation in all cross sections.

Miscoding of ODA vs ODA and IDA defects

Missing IDAs in ODA defects

*Action. Reduce the number of arms required to say IDA is present and discuss in the text OR as previous proposed by HO, that the term: class 1 hallmark defect of the ODA +/- IDA should be used if at all unsure (e.g. a poor sample or unfamiliar sample preparation)

Class 2 defects

3 centres were able to identify the DRC defect

*Action: include MTD in the class 2 defect list

Use of class 2: basal body mislocalisation with few or no cilia –used to describe inadequate samples

*Action: Extend the text around this defect

2.2 2nd validation summary - photograph swap

 100% participation  There were no false positive diagnoses (i.e No non-PCD sample was classed as having a Class1 Hallmark PCD defect) o Normal ultrastructure identified by 18/18 centres  100% correct identification as a class 1 hallmark defect o 18/18 correctly identified MTD + IDA o 17/18 ODA (1 judged as ODA+IDA) o 17/18 ODA+IDA (1 judged ODA- same centre as above (respiratory clinician participant not microscopist or pathologist)  100% correct identification as a class 2 defect o 15/18 central complex defect (3 judged as MTD)

3.0 Link to example images and reports https://uod.box.com/s/3isd4vk26qj2ac738krm2qlnj2gqf6wa 4.0 Table of Genotype by TEM phenotype

Class 1 Gene Comments defects Outer dynein arm defect DNAH5 Can be subtle with some missense mutations DNAI1 DNAI2 DNAL1 NME8 DNAH9 Distal cross sections only CCDC114 ARMC4 CCDC151 TTC25 MNS1 Outer and inner dynein arm DNAAF1 DNAAF2 DNAAF3 DNAAF4 DNAAF5 LRRC6 ZMYND10 SPAG1 C21ORF59 PIH1D3 CCDC103 Inner dynein arm and CCDC39 CCDC40 microtubular disorganisation Class 2 defects

Microtubular disorganisation CCDC164 CCDC65 GAS8 Central complex defect RSPH1 RSPH4A RSPH9 RSPH3 DNAJB13 Some central pair abnormalities and absence of c2b c2b projection Not diagnostic HYDIN absence STK36 Mislocalisation of basal bodies CCNO MCIDAS

Not-diagnostic CCDC11 ENKUR GAS2L2 LRRC56 Can be identified with electron tomography DNAH11 6

5.0 Supplementary Figure 1: Normal ultrastructure in longitudinal section

5.0 Example reports

THE CYPRUS INSTITUTE OF NEUROLOGY AND GENETICS P.O.Box 23462 1683 Nicosia, CYPRUS

Department of Electron Microscopy Tel.: 22392631 (Office) 22392792 (Lab) Fax.: 22358237 EM No.: R-72 (C1) Specimen Request Form

Patient Surname: Name:

ID Number: Date of Birth: Sex:

Patient Address:

Referring Doctor: Hospital File No.:

Site of Biopsy: Nasal Brushing Histology No.:

ELECTRON CRO CO REPORT

The specimen obtained was of very good quality and appeared well preserved. Electron microscopy was

performed on 68 ciliary cross-sections. Ultrastructural analysis revealed abnormal ultrastructure across the

sample (100%) with low numbers of outer and inner dynein arms in all ciliary cross-sections. Among the cross-

sections examined there was limited evidence of tubular disorganization (7%) and central pair disorientation

(9%). Few cross-sections presented with some membrane swelling (6%). Moderate evidence of inflammation and no evidence of bacteria was observed. 3% compound cilia were detected. Summary of key findings: These results are consistent with the diagnosis of Primary Ciliary Dyskinesia, with a class 1 defect. A combined outer and inner dynein arm defect. Date received: 27/06/2018 Date reported: 31/07/2018

The submitted report contains confidential personal data and information and should be protected accordingly. In addition the report or part of the report, should not be provided in any form to third parties.

Name: Example EM No. 18/269 CRN: Date of Reason for

Birth: referral Sample: Nasal brushing

Ciliary profile counts:

Microtubular arrangement Dynein arms

und

Cilia Cilia

arranged Total Total

absent absent

Normal

present

Missing Missing

No arms No

Both Arms Both

Inner Arms Inner

One of Pair of One

Outer Arms Outer

Com

Both of Pair of Both

Other Defect Other

microtubular microtubular arrangement ExtraTubule

Dis Single Tubule Single

83 5 7 5 0 0 0 0 100 29 0 0 0 29

89 3 3 4 0 1 0 0 100 44 0 0 0 44

86% 4% 5% 4.5% - <1% - - 100% - - -

Comments: Unhealthy but adequate sample. Some distorted ciliary membranes. Some ciliary disorientation seen. Normal longitudinal profile.

Summary: Predominantly normal ciliary ultrastructure with both dynein arms present. Normal ultrastructure does not exclude a diagnosis of PCD

International consensus guideline for reporting transmission electron microscopy results in the diagnosis of Primary Ciliary Dyskinesia Shoemark et al.

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